This invention relates to a method for the release of N-glycans and O-glycans (i.e. `N- and O-linked type` oligosaccharides) from glycoconjugates and to isolation of such glycans.
Examples of glycoconjugates are glycoproteins, glycohormones and glycolipids.
An `N-linked type` oligosaccharide is an oligosaccharide which is covalently bonded to a conjugate by an N-glycosidic linkage, and an `O-linked type` oligosaccharide is an oligosaccharide which is bonded to a conjugate by an O-glycosidic linkage. An example of such linkages as typically found on glycoproteins is shown in FIG. 1 of the accompanying Drawings. For the purposes of this disclosure, release refers to an event or process leading to cleavage of the covalent N- and O-glycosidic bonds attaching `N-` and `O-linked-type` oligosaccharides to a conjugate, and isolation refers to an event or process leading to physical separation of released oligosaccharides from conjugate or conjugate-derived materials.
The release and subsequent isolation of `N- and O-linked type` oligosaccharides (hereinafter referred to respectively as N- and O-glycans) from a glycoconjugate are important for several reasons. Principal among these are the following: Firstly, a detailed structural characterization of a glycoconjugate requires structural analysis of any associated glycans. Since several such glycans may be attached to a single conjugate, and since structural analysis of glycans is most accurately performed on single, purified glycans, such an analysis requires prior release of such glycans from the conjugate and subsequent isolation of such glycans from the conjugate, and then purification of glycans from one another. Secondly, such glycans are increasingly recognized as important biological molecules in their own right. A study of the biological properties of N- and O-glycans is preferentially undertaken using N- and O-glycans free of the conjugate. The criteria that ought to be simultaneously satisfied for any successful method for the release and isolation of N- and O-glycans from a glycoconjugate are as follows:
(i) The method of release should preferably cleave the N- and O-glycosidic linkage in a manner independent of the nature of both the oligosaccharide component and the conjugate component. PA1 (ii) The method of release should preferably achieve cleavage without permanent (i.e. not easily reversible) chemical damage to the cleaved glycans. PA1 (iii) The method of isolation should preferably separate N- and O-glycans from a conjugate in a manner independent of the nature of both the oligosaccharide component and the conjugate component, as in (i), above. PA1 (iv) The method of isolation should preferably achieve isolation without causing chemical damage to the cleaved glycans. PA1 (i) The use of alkaline solutions. The N- and O-glycosidic linkages attaching N- and O-glycans to a conjugate are alkali labile, and alkaline solutions can therefore be employed for release of N- and O-glycans. For example, incubation with 1M NaOH at 100.degree. C. for 4 hours has been successfully employed. An established and accepted disadvantage of this method is the alkali-lability of most N- and O-glycans. Most N- and O-glycans that are attached to a peptide are so attached through the structures shown in FIG. 2 of the accompanying Drawings. Upon cleavage (FIG. 2) by alkali, the reducing terminus monosaccharide residue (N-acetylglucosamine or GlcNAc in N-glycans, and N-acetylgalactosamine or GalNAc in O-glycans) is further degraded by the alkali. Such degradation can be prevented by the reduction of the reducing terminus residue by performing the alkali-induced release in the presence of a vast excess of reducing agent. Typically 4M NaBH.sub.4 is used. This reduction involves conversion of the released glycans to the alditol form (FIG. 2). Irrespective, therefore, of the presence or absence of excess reducing agent, the released glycans will be recovered in a permanently chemically altered form (i.e. not as the native compound). This method does not therefore satisfy criterion (ii), above. PA1 (ii) The use of anydrous hydrazine. A few reports in the scientific literature concerning O-glycans suggest that O-glycans may be released from a peptide conjugate after incubating an anhydrous glycoprotein at high temperature (typically 100.degree. C.) for several hours (typically .gtoreq.10 hours) with anhydrous hydrazine. Each such report also indicates that any O-glycans so released are subject to extensive chemical degradation and alteration of the released O-glycans by the hydrazine. Indeed, the general scientific opinion indicates that: `The behavior towards hydrazine of O-glycosidically linked glycans has not yet been elucidated. However some results show that they are profoundly degraded until an alkali-stable linkage stops the `peeling` relaction.` (J. Montreuil et al., in `Carbohydrate Analysis--A Practical Approach`--Ed. M. F. Chaplin and J. F. Kennedy--IRL Press, 1986). With regard to N-glycans, numerous authors have studied the release by hydrazinolysis of N-glycans from glycoproteins. The currently preferred method is that of Rademacher and Dwek (European Patent Application no. 0 215 766A2). This method, addressing only N-glycans, is a method in which a set of conditions were defined allowing recovery of N-glycans in undefined yield, but intact with respect to structure. The method of Rademacher and Dwek was therefore an improvement on all previous methods, but as shown by the results disclosed in this specification, gives a relatively lower yield of N-glycans and would, when applied to gycloproteins containing O-glycans, lead to the recovery of degraded (i.e. chemically altered) O-glycans. These consequences arise from the fact that the study of Rademacher and Dwek did not consider O-glycans (i.e. considered glycoproteins with only N-glycans). Hence this method as currently practiced is not generally applicable to the recovery of intact N- and O-glycans from a glycoprotein sample. PA1 k=reaction rate PA1 A=Arrhenius constant PA1 R=Universal gas constant PA1 E.sub.ACT =Activation energy PA1 T=temperature. PA1 A.sub.ro =Arrhenius constant for release of O-glycans PA1 A.sub.do =Arrhenius constant for degradation of O-glycans PA1 E.sup.R.sub.O =Activation energy for release of O-glycans PA1 E.sub.D.sub.O =Activation energy for degradation of O-glycans PA1 A.sub.rN =Arrhenius constant for the release of O-glycans PA1 A.sub.DN =Arrhenius constant for the degradation of N-glycans PA1 E.sup.A.sub.N =Activation energy for the release of N-glycans PA1 E.sup.D.sub.N =Activation energy for the degradation of N-glycans PA1 R=Universal gas constant PA1 T=Temperature PA1 t=time PA1 (1) Composition analysis of the monosaccharides obtained from the N- and O-glycans. PA1 (2) High-voltage paper electrophoresis and high resolution gel permeation chromatography of the reduced radiolabelled (e.g. tritiated) alditols of the N- and O-glycans.
In addition, it is preferable if the method also achieves recovery in high yield (e.g. .gtoreq.85%) of released N- and O-glycans, irrespective of the amount of starting glycoconjugate.
Previously, two different methods have been used for the release of both N- and O-glycans. These are briefly summarized below and assessed with respect to the above criteria.
A. Enzymatic Methods--For example, the use of proteolytic enzymes such as Pronase.RTM. to obtain glycopeptides from glycoproteins, and/or the use of a mixture of enzymes such as O-glycanase.TM. (E.C. number 3.2.1.97) to cleave some O-glycans from the glycoconjugate and N-glycanase.TM. (E.C. number 3.2.2.18) to cleave some O-glycans from the conjugate. Such methods are generally unsatisfactory for several reasons, but principally because of the selectivity of glycan release. Enzymes are, by their very nature specific, and release only certain glycans and then in a manner influenced by the attached conjugate. That is, enzymatic methods as currently practiced and understood, do not satisfy criterion (i), above.
B. Chemical Methods--Two chemical methods have been practiced for the release of N- and O-glycans. These are:
In summary, none of the above mentioned methods as currently practiced and understood is suitable for the release of intact, unaltered, N- and O-glycans.